Molecularly imprinted polymers selective for nitrosamines and methods of using the same

ABSTRACT

A class of molecularly imprinted polymers that specifically recognizes and binds to nitrosamines members of which class are useful, for example, in analysis and separation of nitrosamines from biological fluids. Such molecularly imprinted polymers are also useful in methods of treating and manufacturing tobacco products and materials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/604,003, filed Nov. 21, 2006, which is a continuation ofinternational PCT application Serial No. PCT/SE2005/00773 filed May 24,2005 and published as WO2005/112670 in English on Dec. 1, 2005, whichclaims priority under 35 U.S.C. §119 to U.S. provisional applicationSer. No. 60/573,337 filed May 24, 2004. The entire contents of theaforementioned applications are herein expressly incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to a class of molecularly imprintedpolymers and use of the molecularly imprinted polymers in bioanalysisand separation of nicotine metabolites. The invention further relates tomethods of using the molecularly imprinted polymers to treat tobacco,tobacco substitutes, and their derivatives to reduce the level oftargeted compounds therein.

BACKGROUND OF THE INVENTION

In the fields of medical, dietary, environmental and chemical sciencesthere is an increasing need for the selective separation of specificsubstances from complex mixtures of related substances. The aim can bethe quantitative extraction of a certain compound or compounds, themeasurement of their concentration or the selective removal of a targetcompound from a multi-component mixture.

Stricter health controls have increased the demand for methods allowingsensitive and selective quantification of hazardous products andmetabolites from certain everyday substances in widespread use. Ofparticular concern are chemical compounds related to use oftobacco-based products, which compounds are either originally present inthe raw tobacco leaf itself or generated during the smoking process.Nitroso-containing compounds, such as nitrosamines, are regarded asbeing of special significance in this regard.

With the aim of reducing the occurrence of hazards related to smoking,certain pharmaceutical products have been produced containing only theneuroactive substance, nicotine, the chemical claimed to be responsiblefor the dependence aspects of smokable material.

Among the nicotine formulations for smoking cessation therapy, nicotinechewing gum has found the most widespread use. The quality controlrequired during production includes monitoring of the nicotine level (2or 4 mg per gum) as well as monitoring of the primary nicotine oxidationproducts cotinine, myosmine, nicotine-cis-N-oxide,nicotine-trans-N-oxide and beta-nicotyrine. Quantitation of nomicotine,anatabine and anabasine is also desirable, if not required. According tothe United States Pharmacopeia (U.S.P.) the gum formulation shouldcontain between 95% and 110% of the amount of nicotine given on thelabel and the amount of each oxidation product should not exceed 0.1% ofthe nicotine amount.

Despite the use of such cigarette substitutes, nitrosamine nicotinemetabolites may be produced in vivo by natural metabolic processesduring the residence of the nicotine within body tissues. The levels ofthese metabolites remain below the concentrations at which mostanalytical procedures can perform quantitatively. The need for methodswhich are capable of monitoring these levels, as well as the levels ofother nicotine metabolites, is therefore of importance. Typically, suchmonitoring is performed on human urine samples in which levels of suchsuspected carcinogens are extremely low.

Targeted compounds for quantification, reduction or removal from tobaccoor smoke are known and include the major components of tobacco-specificnitrosamines (TSNAs) and their alkaloid precursors: NNK,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNA,4-(methylnitrosamino)-4-(3-pyridyl)butanal; NNN, N-nitrosonomicotine;NAB, N-nitrosoanabasine; NAT, N-nitrosoanatabine; NNAL,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; iso-NNAL,4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol; iso-5 NNAC,4-(methylnitrosamino)-4-(3-pyridyl)butanoic acid.

To properly quantify how much of such targeted compounds are present inhuman biological fluids, methods are being developed to analyse thealkaloids, especially the nitrosylated decomposition products andmetabolites in tobacco. Existing chromatographic separation orextraction methods used for this analysis lack the robustness,sensitivity and speed required in order to handle the large number ofsamples generated when screening the general population. With existingmethods, the low concentration of the nitrosamines, which are typicallypresent in picograms per milliliter, demands extensive samplepreparation with multi-step extractions and often chemicalderivatization (for example deuteration prior to mass spectrometry) ofthe analyte prior to analysis. One reason for this complexity is thatthe existing separation materials are not selective as, for example, anantibody or biological receptor might be for the metabolites in questionbut rather rely on physico-chemical properties like charge orhydrophobicity of the metabolites for the separation behaviour. Thesephysico-chemical properties may be shared by many other irrelevantmolecules in the sample.

A typical procedure might involve up to seven work-up steps includingcentrifugations, pH adjustments, enzymatic treatments, etc., which maysum up to a preparation time of many hours or even days per sample. Withsuch cumbersome procedures, loss of material during the process can leadto errors in estimation of the original sample concentrations, requiringextrapolation back from the final measurement, rather then reliance ondirect measurement, to obtain the original concentration in the sample.A quick and simple method for the analysis of tobacco-specificnitrosamines is therefore a significant unmet medical analytical need.(See, e.g. Byrd & Ogden, Journal of Mass Spectrometry, 2003, 38, 98-107and Wu et al. Anal. Chem. 2003, 75, 4827-4832).

During recent years numerous reports of selective recognition of smallmolecules with materials prepared by molecular imprinting (molecularlyimprinted polymers or MIPs) have appeared. See, for example, Wulff, G.Angew, Chemie. Int. Ed. Engl. 1995 (34) 1812. MIPs are polymers havingreactive sites adapted to bind selectively with targeted compounds.Non-covalently prepared molecularly imprinted materials have been usedfor chiral recognition of a variety of small molecules includingtherapeutic drugs, sugars, molecular bases, and pesticides as well assteroid and peptide hormones. Examples of the same are described in, forexample, Sellergren, B. Trends Anal. Chem. 1997 (16) 310. The highaffinity and selectively for the target analyte exhibited by some of theimprinted materials have justified a comparison with the correspondingimmuno-affinity (IA) phases. In contrast to the latter phases however,the MIP materials are straightforward to prepare, stable in most mediaand reusable over long periods of time. Applications of the MIPmaterials in chromatography, separation (continuous or batch), chemicalsensing or in specific assays are therefore under investigation.

Another application is solid-phase extraction (SPE, see Mayes, A. G.,Mosbach, K. Trends Anal. Chem. 1997, 16, 321) of analytes present in lowconcentrations in biological samples, or in complex matrices. SPE maylead to selective enrichment and clean-up of an analyte to levels notachievable with existing methods. Molecularly imprinted solid phaseextractions (MISPE) have been used in bioanalysis, food analysis andenvironmental analysis. In these examples selective enrichment andclean-up of the analyte is obtained resulting in higher accuracy and alowering of the detection limit (LOD) in the subsequent chromatographic(eg HPLC) or mass spectrometric quantification.

In view of their high selectivity combines with good affinity for thetarget molecule or a group of target molecules, MIPs have attractedconsiderable interest from the food industry as a tool to improve foodquality. This requires the use of a MIP for selective removal ofundesirable components from the food matrix. Since these components areoften present in low concentrations, the saturation capacity of the MIPis typically not a limiting factor.

The preferred specifically designed MIP material of the invention iscapable of selectively absorbing the most common nitrosylated nicotinederivatives from complex matrices, such as urine, giving quantitativerecovery and thereby leading to low errors in the estimation of suchhazardous chemical concentrations.

In addition to quantifications it is also well known to attempt toreduce the harmful effects of consuming material containing tobacco,tobacco substitutes or mixtures thereof by reducing the levels oftargeted compounds. Such reductions can be made in the material itselfor in a derivative thereof such as an extract of the material. Reductioncan also be effected in the thermal decomposition products of thematerial, i.e. mainstream and sidestream smoke obtained by combustion,or the aerosols produced by heating the material to a temperature belowits combustion temperature.

One very well known method for this sort of reduction is to contact thethermal decomposition products of the material with a filter thatadsorbs undesired components therefrom. An alternative method involvessolvent extraction of the material, for example as disclosed in thespecification of U.S. Pat. No. 5,601,097. According to thatspecification, 5 the protein content of tobacco material is reduced bytreating the tobacco with a solution containing a surfactant to extractpolypeptides, separating the solution, removing the surfactant and thepolypeptides from the solution, and recombining the solution with thetobacco material. International patent application specification WO01/65954 discloses a process in which tobacco is contacted with asupercritical extraction fluid such as supercritical carbon dioxide toselectively reduce or eliminate nitrosamines.

These processes are equally applicable to both tobacco itself and totobacco substitutes i.e. natural or synthetic materials having similarcharacteristics to natural tobacco that enable them to be consumed in asimilar way to tobacco, whether by smoking, chewing, inhalation orotherwise.

There has been an attempt to remove nicotine from tobacco smoke usingMIPs, as reported in Liu, Y., et al., Molecularly imprinted Solid-PhaseExtraction Sorbent for Removal of Nicotine from Tobacco Smoke,Analytical Letters, Vol. 36, No. 8, pp 1631-1645 (2003). The MIPdescribed in the article was designed to bind nicotine and not the moretoxic nicotine metabolites such as nitrosamines. It is unclear if theMIP was in fact selective for nicotine as the scientific methodproducing the data was lacking in key control-checking elements.

Therefore, there remains a need in the art for novel MIPs and methods ofemploying the same, particularly in the field of nicotine and nicotinemetabolites.

SUMMARY OF THE INVENTION

Broadly, the present invention provides a molecularly imprinted polymer(MIP) selective for nitroso-containing compounds.

The preferred MIPs of the invention are selective for nitrosamines, inparticular TSNAs or the volatile nitrosamines found in the vapor phaseof the thermal decomposition products of smoking materials. Anotherpreferred MIP of the invention is selective for one or more of thenitrosylated derivatives of nicotine or the other alkaloids found intobacco, namely nornicotine, anabasine and anatabine.

The MIPs of the invention can be obtained, for example, byco-polymerising a functional monomer, or monomers and a cross-linker inthe presence of a structural analogue of a nitrosamine, in apolymerization medium containing a free radical initiator, after whichthe template is removed from the MIP.

The invention includes the use of the molecularly-imprinted polymers ofthe invention for analytical and preparative extractions, inchromatography, for analytical sample pre-treatment, in chemical sensorsor as a solid phase filter for extraction of nicotine nitrosamines fromnicotine-containing substances or devices.

Additionally, the invention includes a method of reducing the level of atargeted component in a tobacco product, in which the tobacco product istreated with a MIP which is selective for at least onenitroso-containing compound. Further, the invention provides methods ofmanufacturing a smoking material which incorporates use of MIPs toselectively remove nitroso-containing compounds.

The present invention includes the treatment of tobacco products withMIPs to reduce the level of nitroso-containing compounds therein.

In this specification, “tobacco product” means a material containingtobacco (including tobacco leaf or tobacco stem), or a tobaccosubstitute, or a blend of tobacco and tobacco substitutes, andderivatives of such material, including extracts of the material, smokeproduced by thermal decomposition of the material and aerosols producedby heating the material to below its combustion temperature.

Where the tobacco product is a derivative produced by the thermaldecomposition of material containing tobacco or a tobacco substitute,the decomposition may be effected by combustion of the material, as in aconventional cigarette, or by heating the material to a temperaturebelow its combustion temperature, in accordance with a process used insome known alternative tobacco products in order to produce an aerosolthat is inhaled by the consumer.

Alternatively, the tobacco product may be a derivative produced bycontacting material containing tobacco or a tobacco substitute with asolvent. In particular, the invention provides a method of manufacturinga material for smoking comprising the steps of extracting smokablematerial with a solvent, treating the extract with a molecularlyimprinted polymer selective for at least one nitroso-compound to reducethe level thereof in the extract and combining the treated extract withthe smokable material.

In this process, the smokable material may be in any convenient form,for example fines, stems, scraps, cut lamina, shredded stems, or anycombination thereof. The solvent may be aqueous or non-aqueous, such asmethanol, ethanol or a super-critical fluid extraction medium, such assuper-critical carbon dioxide liquid. The extraction may be carried outunder any conditions favoring the extraction of nitrogen-containingcompounds from tobacco.

The invention also includes a smoking article comprising tobacco ortobacco substitute, and a molecularly imprinted polymer selective forthe removal of at least one nitroso-containing compound from the thermaldecompositions product thereof.

The smoking article of the invention may take any conventional form, forexample a cigarette, cigar or cigarillo. In particular the smokingarticle may comprise a rod of smoking material optionally in a wrapper,with or without a filter. The wrapper may be of paper, tobacco leaf,reconstituted tobacco or a tobacco substitute. Alternatively, where, forexample, the smoking article is intended to produce low emissions ofsidestream smoke, or lower levels of pyrolysis products in themainstream smoke, the wrapper may be composed of non-combustibleinorganic material such as a ceramic material. The filter may be of anysuitable material, for example fibrous cellulose acetate, polypropyleneor polyethylene, or paper.

The smoking material is preferably tobacco but may be a tobaccosubstitute such as non-tobacco smoking material. Examples of non-tobaccosmoking materials are dried and cured vegetable material, includingfruit materials, and a synthetic smoking material such as may beproduced from alginates and an aerosol-generating substance such asglycerol. The smoking material may also comprise a blend of tobacco andnon-tobacco smoking materials. Where the smoking material comprisestobacco, the tobacco may of any suitable type, or a blend thereof,including air-cured, fire-cured, flue-cured, or sun-cured lamina orstem, and may have been processed using any appropriate process. Forexample, the tobacco may be cut, shredded, expanded or reconstituted.The smoking material may also include conventional additives, such asameliorants, colorants, humectants (such as glycerol and propyleneglycol), inert fillers (such as chalk), and flavourings (such as sugar,liquorice and cocoa).

The invention may also be applied to tobacco that is intended for oralor nasal consumption by sucking, chewing, or nasal ingestion, ratherthan smoking. Such products include snuff, snus and “hard” or chewingtobacco.

The molecularly imprinted material may be incorporated is the smokablematerial. Accordingly, the invention includes smoking materialcontaining a molecularly imprinted polymer selective for the removal ofat least one nitroso-containing compound from the thermal decompositionproducts of the smokable material.

Alternatively, where the smoking article comprises a rod of smokablematerial is a wrapper, the molecularly imprinted material may beincorporated in the wrapper. The invention therefore includes wrappermaterial for smoking articles comprising a molecularly-imprinted,polymer selective for the removal of a targeted component from thethermal decomposition products of a smoking material. The wrapper may bea cellulose-based material such a paper or a tobacco based material suchas reconstituted tobacco.

The preferred smoking articles of the invention are cigarettes,comprising a rod of tobacco, wrapper, and a filter including amolecularly imprinted polymer selective for the removal of at least onenitroso-containing compound from the thermal decomposition products of asmokable material.

The invention also includes a smoke filter comprising a molecularlyimprinted polymer selective for the removal of at least onenitroso-containing compound from the thermal decomposition products of asmoking material. The smoke filter may be produced separately from thesmoking article, for example in the form of a cigarette or cigar holder,or it may be integrated into the smoking article, for example in theform of a cigarette with a filler tip.

Smoke filters in the form of filter tips may be of any conventionalconstruction. For example it may in the form of a “dalmation” typefilter comprising a section of fibrous filter material, such ascellulose acetate, the molecularly imprinted polymer being in particularform and distributed throughout the section. Alternatively the filtermay be in the form of a “cavity” type filter, comprising multiplesections wherein the molecularly imprinted polymer may lie between twoadjacent sections of fibrous filter material. The smoke filter may alsocomprise other adsorbent materials such as an ion-exchange resin, azeolite, silica, alumina or amberlite.

In use, the smoke passes through the filter, the molecularly imprintedpolymer selectively adsorbs and retains the targeted compounds from thesmoke and the filtered smoke is delivered to the smoker.

The smoke filters and smoking articles according to the invention mayinclude means for protecting the molecularly imprinted polymer from, orreducing its exposure to, smoke when in use. This may be achieved in anumber of different ways. For example the smoke filter may comprise afilter element for adsorbing materials from the vapor or particulatephase of smoke. Such filter elements may comprise a general adsorbentsuch as activated carbon, which may be in any convenient form, such asthreads, particles, granules, cloth, or paper. The filter element mayalso be a selective adsorbant such as an ion-exchange resin, a zeolite,silica, alumina or amerlite. The means for protecting the catalyst mayinclude two or more such filter element of different compositions, forexample a first filter element of cellulose acetate, and a second filterelement of activated carbon. The provision of multiple filter elementsin smoke filters and smoking articles is well known, and anyconventional configuration of filter, and associated methods ofconstruction, may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an outline of the procedure for synthesis of an imprintedpolymer;

FIG. 2 shows the nitrosamine functional group and examples of nicotinerelated-nitrosamine targets;

FIG. 3 shows isosteric analougues of nitrosamines;

FIG. 4A shows examples of amide and sulfonamide based target analogs;

FIG. 4B shows an enamine target analogue (MPAPB) used as a template toprepare a MIP for extraction of NNAL;

FIG. 4C shows pyridine carbinol used as a template prepare a MIP forextraction of NNAL;

FIGS. 5A and 5B show recovery rates of NNAL using an NNAL-selective MIP;

FIG. 6 shows chromatograms obtained after analysis of 1 mL human urinespiked with 0.25 μg NNAL (represented by solid line) and 1 mL blankhuman urine (represented by bold line);

FIG. 7 show an overlay of chromatograms obtained after sample analysisin the presence of nicotine where the solid line represents NNAL andnicotine-spiked sample, dashed line represents eluent collected fromloading 1 mL of the NNAL and nicotine-spiked sample, and the long dashedline represents a wash of (NH₄)H₂PO₄, pH 4.5;

FIG. 8 is a side elevation, partly longitudinal cross-section andpartially broken away view of a smoking article with a smoke filteraccording to the invention;

FIG. 9 is a similar view to FIG. 8 of a smoking article with analternative smoke filter according to the invention;

FIG. 10 shows the chemical structure and boiling point for threeproducts described in Example 6; and

FIG. 11 shows the chemical structure and boiling point for selectvolatile nitrosamines.

In the drawings, similar features are gives like reference numerals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Molecular imprinting typically consists of the following steps: (1) atemplate compound, which may be the targeted molecule or a structuralanalogue thereof, is allowed to interact with a selected functionalmonomer, or monomers, in solution to form a template-monomer complex;(2) the template-monomer complex is co-polymerized with a cross-linkingmonomer resulting in a polymeric matrix incorporating the templatecompound; (3) the template compound is extracted from the polymer matrixto form a MIP that can be used for selective binding of the targetedmolecule. Prior to step (3), where the MIP is prepared as a solidpolymer (or monolith) it is typically crushed and sieved to obtain adesired size fraction of particulate material. When prepared by eithersuspension or emulsion polymerization methods, such crushing and sievingis unnecessary since the particle size can be controlled within thedesired limits during the polymerization process. Particulate materialprepared by any of the aforementioned methods can be packed into achromatographic or solid phase extraction column and used forchromatographic separation of the template from other components of amixture, including molecules with similar structures or functionalities.

The reactive sites on the molecularly imprinted polymer exposed byremoval of the template composed will be in a stereo-chemicalconfiguration appropriate for reaction with fresh molecules of thetargeted molecule. As a result, the polymer can be used for selectivebinding of the targeted molecule.

Currently the most widely applied technique to generate molecularlyimprinted binding sites is via the ‘non-covalent’ roots. This makes useof non-covalent self-assembly of the template compound and functionalmonomers to form the template-monomer complex, followed by free radicalpolymerization in the preserve of a cross-linking monomer and finallytemplate compound extraction. Covalent imprinting, in which the templatemolecule and a suitable monomer or monomers are covalently boundtogether prior to polymerization can also be carried out according toknown methods. The birthing properties of the MIPs formed by either ofthe above methods can be examined by reminding of the template molecule.

The polymerization is performed in the presence of a pore-formingsolvent called a porogen. In order to stabilize the electrostaticinteractions between the functional momomers and the template compoundthe porogen is often chosen from among aprotic solvents of low tomoderate polarity. Ideally, template compounds exhibit moderate to highsolubility in the polymerization media and these, or their structuralanalogues, can therefore be used directly using this standard procedure.

While it is possible to use the targeted molecule itself as thetemplate, a structural analog of the target molecule is commonlypreferred because: (a) the targeted molecule may be unstable under thepolymerization conditions or may inhibit the polymerization; (b) thetargeted molecule may not be available in sufficient quantities due tocomplexity of its synthesis or cost, or both; (c) the template may beinsoluble or poorly soluble in the pre-polymerization mixture; (d) theMIP may remain contaminated by low levels of the targeted moleculeretained in poorly accessible regions of the polymer matrix, which maybleed from the MIP during use; and/or (e) the target analyte(s) maypresent a significant health risk and should not be used as atemplate(s).

In the case of nitroso-compounds, particularly the compounds known asTSNAs described below, it is often more convenient to use functionalanalogues thereof as template compounds. For example, glucosederivatives of TSNAs may be particularly useful as template compounds,see FIG. 2.

Where the MIP is derived using a functional analog of the targetedcompound, the functional analogue should be isosteric and preferablyalso isoelectronic with the targeted compound, or it may contain asubstructure of the targeted compound where strong interactions may belikely.

Nitroso-containing compounds, particularly nitrosamines, which have thegeneral formula O═N—N(R₁)(R₂) are among the numerous ingredients oftobacco and tobacco smoke that have been suggested as having a harmfuleffect on consumers.

One particular class of nitroso compounds to which the present inventionis applicable is the group of nitrosamines that occur naturally intobacco, known as tobacco-specific nitrosamines (TSNAs), which arederived from the alkaloids that occur naturally in tobacco namelynicotine, nornicotine, anabasine and anatabine. TSNAs include:

In addition, a group of compounds known as volatile nitrosoamines isfound in the vapor phase of tobacco smoke. This group includes thefollowing compounds:

Other nitroso-containing compounds have also been identified in chemicalstudies of tobacco or tobacco smoke, for example:

Possible isosteric analogs for the targeting of nitrosamines are seen inFIG. 3. The molecules shown are all derivatives of the parent amine andcan be synthesized in a single step from the secondary amine andcorresponding aldehyde or acid chloride. Molecular models of the enamine(FIG. 4A) have shown a good steric complementarily with one of thenitrosamines of interest, NNAL.

During the design of a suitable template compound for the target analyteNNAL, a particularly interesting template was identified, correspondingto the pyridine carbinol substructure but surprisingly lacking thenitrosamine moiety (FIG. 4B). If sufficient binding affinity andselectivity can be obtained for sub-structural templates, this is thepreferred approach. In fact, the binding affinity, selectivity andrecoveries obtained with this pyridine carbinol MIP are superior to theMIPs obtained, with the more complex enamine template. Thus, theinvention provides a surprisingly effective MIP which comprises a simpletemplate lacking certain key features of the target but providing foreffective binding with those target nitrosamines which contain thepyridine-methanol moiety.

Using the functional monomer methacrylic acid (MAA), either of twocrosslinkers, ethylene glycol dimethacrylate (EDMA) or trimethylopropanetrimethacrylate (TRIM) and either of the two NNAL analogs,4-(Methylpropenyl-amino)-1-pyridin-3-yl-butan-1-ol (4MPAPB, FIG. 4A) andpyridine carbinol (FIG. 4B) as templates, two different polymers areobtained both exhibiting strong affinity and selectivity for NNAL inorganic and aqueous solvent environments.

This invention includes an extraction method for quantitative recoveryof the nicotine analog NNAL that entails the steps of preparation of anNNAL-selective MIP in a chromatographic material format, columnconditioning, application of a urine sample, removal of interferingcompounds and finally selective elution of the NNAL analyte.

By way of explanation and not of limitation, the invention will befurther described in more detail with reference to a number of examples.The invention refers to template molecules, polymer materials designedto bind nitrosamines deriving from nicotine and present in organic oraqueous systems, and finally use of said materials in, for example,analytical or preparative separations, in chromatography, for analyticalsample pre-treatment and in chemical sensors.

Unless otherwise described, materials are commercially available or canbe prepared by conventional techniques. See, for example, B. Sellergren(Ed.) Molecularly-imprinted Polymers: Man made mimics of antibodies andtheir application in analytical chemistry, part of the series Techniquesand Instrumentation in Analytical Chemistry, Elsevier Science,Amsterdam, Netherlands, 2001.

Example 1: Synthesis of Enamine Template (MPAPB)

Anhydrous toluene (freshly dried over sodium) 2 ml was added to a vialcontaining 4-methylamine-1-(3-pyridyl)-1-butanol (100 mg). 500 mgfreshly dried Molecular sieve was added to it. The mixture was stirredfor 1 hour under N₂. To the mixture 100 μl propionaldehyde was added.The mixture was stirred at 55° C. for 4 hours. The reaction wasmonitored by HPLC after 1.5 hours. The color of the product wasorange-yellow in toluene. The crude product in toluene was directly usedfor the synthesis of the MIP alter filtration without publication.Template MPAPB yield was around 90%.

Example 2: Synthesis of MIP Using Pyridine Carbinol as Template

To pyridine methanol (97 μl) 3.74 ml of purified TRIM (purified overbasic alumina), functional monomer MAA (1020 μl), porogenic solventtoluene (7.1 ml) and finally initiator ABDV (63 mg) were added andstirred until a clear solution was obtained. The solution wastransferred to a glass vial, purged with nitrogen for 5 minutes andflame sealed. Heal induced polymerization was carried out at 45° C. for24 hours. The polymer mixture was then cured at 70° C. for a further 24hours.

Processing of the crude MIP material was as follows: the MIP wascoarsely crashed and transferred to a Soxhlet thimble. It wasexcessively washed first with methanol for 12 hours and then with aceticacid for 12 hours in order to remove any remaining template and othernon-reacted monomers. After these first extraction steps, the polymerwas vacuum dried and then ground and sieved to a line powder within asize range of 20 to 90 μm. As a final extraction step, the finely groundMIP was subjected to a 40 minutes microwave assisted solvent extractionusing formic acid as the extraction solution. After drying, the MIP wasready for use.

Example 3: Use of MIPs as Selective Sorbents in SPE

In one embodiment of the invention, the MIP can be packed into solidphase extraction columns for the selective extraction of NNAL from abiological matrix. First, a polypropylene frit was placed in anappropriate SPE column (typically 10 ml capacity for analytical uses),25 mg of the MIP was then added on top to form a MIP bed and the secondfrit was firmly pressed onto the surface of the MIP bed. Conditioning ofthe column was carried out in the following order: 1 ml DCM, 1 ml MeOHand finally 1 ml distilled water were added to the MIPSPE.

The sample, e.g. human urine (5 mL) containing low amounts of theanalyte was allowed to pass through the conditioned MIPSPE column. Thecolumn was then subjected to vacuum in order to remove the water untilthe material was dry. Then, polar interfering substances that may havenon-specifically associated with the MIP were eluted by a wash with 1 mldistilled water. Again, a drying step using several minutes of vacuumwas performed in order to enable the so-called phase-switch (change ofthe environment from aqueous to organic). At this point, non-polarinterfering substances were removed by washes with each 1 ml toluene,toluene:DCM (9:1) and toluene:DCM (4:1). The final selective elution ofNNAL was carried out in 3 times elution steps, each of 1 ml DCM.

After solvent evaporation, the samples were reconstituted in the mobilephase and analyzed on an HPLC system: e.g. Merck-Hitachi (L-7000 system)using a beta-basic C18 column, 5 μm, 150×2.1 mm+pre-column 10×2.1 mm.Flow was at 0.25 mL/min, injection volume 100 μL, temperature 30° C. anddetection at UV 262 nm. The mobile phase consists of 50 mM NH4PO4 pH 3,5 mM octanesulfonic acid 20% methanol.

Under these conditions, NNAL was obtained as a clearly distinguishabledouble peak eluting at about 8-10 minutes (see FIG. 6, where a 1 mlsample of human urine spiked with 0.25 μg NNAL is compared withNNAL-free urine). The double peak is characteristic for NNAL as itcorresponds to its two rotamers. From the structure of NNAL, it can bedemonstrated that the side chain on the pyridine ring can have differentconformational states. The preferred conformations are called rotamersand for NNAL there are two major conformations. The retention of thesetwo rotamers on an HPLC column will differ. As shown in FIG. 6, the NNALpeak is cleanly separated from interfering substances. It can thereforebe easily and accurately quantified. Recovery rates for NNAL (defined asAmount recovered/Amount loaded×100) are typically up to 90%, dependingon the initial levels of NNAL in the biological sample. Recovery ratesof close to 100% have been seen with samples containing 50 pg/ml and 500pg/ml of NNAL (FIGS. 5A and 5B).

Example 4: Use of MIPs as Selective Sorbents in SPE in the Presence ofNicotine

Another application of the invention is the use of the MIP as aselective sorbent for NNAL where there are high levels of nicotinepresent. This illustrates the wide scope of applications of the MIPmaterial and how the selective nature of the MIP can be finetuned forparticular samples.

SPE columns were prepared as described in Example 3. Conditioning of theSPE column was carried out in the following order: 1 ml DCM followed by1 ml MeOH followed by 1 ml 50 mM (NH₄)H₄PO₄, pH 4.5. The sample, in thisexample 5 mL human urine-containing low amounts of the analyte wasallowed to pass through the conditioned MIPSPE column. The column wasthen subjected to a mild vacuum (e.g., 10-80 kPa) to remove water untilthe material was dry. Polar interfering substances that may havenonspecifically associated with the MIP were eluted by a wash with 1 ml50 mM (NH₄)H₂PO₄, pH 4.5. Another drying step of several minutes of mildvacuum was performed. Further, washes with 1 ml each toluene,toluene:DCM (9:1) and toluene:DCM (4:1) were performed in that order.The final selective elution of NNAL was carried out in 3 elution stepseach of 1 ml DCM.

After solvent evaporation the samples were reconstituted in the mobilephase and analyzed on an HPLC system similar to that described inExample 3. An example chromatogram is shown in FIG. 7, which illustrateshow the NNAL is selectively retained on the MIP while the nicotine isremoved in the buffer wash.

Example 5: Smoking Articles Incorporating MIPs

Referring to the drawings, FIGS. 8 and 9 illustrate smoking articles inthe form of cigarettes having a rod 1 of tobacco encased in a wrapper 2attached to a smoke filter 3 by means of a tipping paper 4. For clarity,the tipping paper 4 is shown spaced from the wrapper 2, but in fact theywill lie in close contact.

In FIG. 8, the smoke filter 3 comprises three cylindrical filterelements 3 a, 3 b, 3 c. The first filter element 3 a at the mouth end ofthe filter is 7 mm in length, composed of cellulose acetate towimpregnated with 7% by weight of triacetin plasticizer having a 25 mmwater gauge pressure drop over its length. The second filter element 3b, positioned centrally is a cavity 5 mm in length containing 150 mg ofactivated carbon granules. The third filter element 3 c adjacent the rod1 is 15 mm in length, has a 90 mm water gauge pressure drop over itslength, and comprises 80 mg cellulose acetate tow. The tow isimpregnated with 4% by weight of triacetin and has 80 mg of MIP specificfor volatile nitroamines, produced as described in Example 6 below,distributed evenly throughout its volume in a “Dalmatian” style.

The cigarette shown in FIG. 9 is similar to that of FIG. 8 except thatthe smoke filter 3 has four coaxial, cylindrical filter elements 3 a, 3b, 3 c and 3 d. The first filter element 3 a at the month end of thecigarette is 5 mm in length, and composed of cellulose acetate towimpregnated with 7% by weight of triacetin plasticizer. The secondfilter element 3 b, positioned adjacent the first filter element 3 a isa cavity 5 mm in length containing 200 mg of molecularly-imprintedpolymer specific for volatile nitrosamines, produced as described inExample 6 below. The third filter element 3 c adjacent the second filterelement 3 b is 10 mm in length and comprises cellulose acetate towimpregnated with 7% by weight of triacetin. The fourth filter element 3d lies between the third filter element 3 c, is 7 mm in length andcomprises 80 mg of granular activated carbon. A ring of ventilationholes 5 is formed in the tipping paper 4 in a radial plane A-A whichdeliver air into the third filter element 3 c about 3 mm downstream ofthe junction with the fourth filter 5 element 3 d when smoke is inhaledthrough the cigarette.

The following Examples further illustrative this aspect of theinvention.

Two equivalents of an appropriate secondary amine, e.g. dimethylamine,diethyl amine, pyrrolidine, piperidine or morpholine, are dissolved inanhydrous ether and freshly dried molecular sieves (50 g/mole amine) areadded. The mixture is then cooled to −5° C. and stirred. One equivalentof propionaldehyde is then added drop-wise to the cooled mixture,maintaining the temperature at 0±5° C. The mixture is allowed to standin a cold bath overnight and is then filtered. The product is obtainedin approximately 50% yield by distillation of the filtrate under reducedpressure, depending on the boiling point of the product. By way ofexample, structures and boiling points are shown in FIG. 10. (See,Brannock, et. al., J. Org. Chem., 1964, 29, 801-812.)

By using a strong acid functional monomer, the enamine is protonated,thus creating the necessary non-covalent interaction during theimprinting step. The positive charge resides on the carbon atom attachedto the nitrogen, a structure stabilized due to derealization to give animinium ion. This positions the acidic functional monomers correctly forlater recognition of volatile nitrosamines. As there is no opportunityto delocalise the positive charge, protonation of the enamine nitrogenis disfavored. (See, Cook, et al., J. Org. Chem., 1995, 60, 3169-3171.)

It may be preferred to use a more strongly-acidic functional monomerthan MAA. Further 5 embodiments incorporate 4-vinylbenzoic acid or4-vinyl benzene sulphonic acid as functional monomers.

Example 7: Synthesis of a MIP Using an Enamine as Template

A pre-polymerization solution is prepared by dissolving the desiredenamine (1 mmol), an acidic functional monomer (4 mmol), a cross-linkingmonomer (20 mmol) and a free-radical initiator (1% w/w total monomers)in a appropriate porogenic solvent. The functional monomer is either MAAor trifluoromethacrylic acid (TFMAA), the cross-linker is either EDMA orTRIM, the free-radical initiator is ABDV and the porogenic solvent isone of chloroform, toluene, acetonitrile or acetonitrile/toluene (1/1v/v). The solution is transferred to a polymerization vessel, cooled to0° C. and then purged with N₂ for 5 minutes, after which the vessel isflame sealed. Polymerization is initiated at 45° C. and allowed tocontinue at this temperature for 24 hours. The polymer is then cured at70° C. for a further 24 hours.

The crude MIP material is then processed. The MIP is coarsely crushedand transferred to a Soxhlet thimble. It is then extensively extracted(i) with methanol for 12 hours and (ii) with acetic acid for 12 hours,in order to remove the template molecule and any unreacted monomers.After these first extraction steps, the polymer is vacuum dried, ground,and sieved to give particles of the desired size range, e.g. 25-36 μm.The finely-ground MIP is then subjected to a final extraction step,involving 40 minutes microwave assisted extraction using formic acid asthe extraction solvent. The MIP is then dried in vacuo for 24 hours.

Alternatively, the target TSNA may be used in place of the enamine. Theboiling points of select volatile nitrosamines at normal atmosphericpressure are shown in FIG. 11.

Example 8: Use of the MIP Material of Example 2 and/or Example 7 in theTreatment of Tobacco Extracts

The polymer produced in accordance with the method of Example 2 orExample 7 is incorporated into a solid phase extraction column, and thecolumn is conditioned by passing through dichloromethane (DCM), methanoland finally distilled water.

Shredded Burley tobacco leaf is extracted with water for 15 minutes at60° C. The tobacco is separated from the solution by filtration anddried. The solution is passed through the column and allowed to adsorbTSNA from the extract. The column is then drained and the solutionconcentrated by film evaporation, the concentrate is then recombinedwith the extracted tobacco and dried in air.

TSNA adsorbed by the polymer can be eluted from the column using DCM.

Example 9: Use of the MIP Material of Example 2 or Example 7 in theTreatment of Tobacco Extracts

Flue-cured shredded tobacco leaf is extracted with water for 15 minutesat 60° C. The tobacco is separated from the solution by filtration anddried. The solution is mixed with the MIP of Example 2 or Example 7,during which period the polymer adsorbs the TSNAs selectively from thesolution. The MIP is then mechanically separated from the extract byfiltration or by centrifugation. The solution is concentrated byevaporation; the concentrate is then recombined with the extractedtobacco and dried in air.

The MIP can be regenerated by elution with DCM, methanol and finallydeionised water or pH 4 buffer, for reuse.

Example 10: Use of the MIP Material of Example 2 or Example 7 in theTreatment of Tobacco Extracts

Using a continuous extraction process, US Blend-type shredded tobaccoleaf is loaded into a first extraction chamber into which super-criticalcarbon dioxide is fed. After contacting the tobacco, the carbon dioxideis fed into a second extraction chamber containing a MIP produced asdescribed in Example 2 or Example 7. Having contacted the polymer, thecarbon dioxide is returned to the first extraction chamber and contactedagain with the tobacco. The cyclic process is continued until the TSNAcontent of the tobacco has been reduced to a desired level, whereuponthe carbon dioxide is vented from the system, and the tobacco removedfrom the first chamber. The MIP in the second chamber is thenregenerated using DCM, methanol and acetic acid.

Example 11: Use of the Molecularly-Imprinted Polymer Material Developedfor 4-methylnitrosoamino-1-(3-pyridyl)-1-butanol (NNAL), in theTreatment of an NNAL and Nicotine Containing Solution

The polymer produced in accordance with the method of Example 2 wasincorporated into a solid phase extraction column, and the column wasconditioned by passing through phosphate buffer solution.

Aqueous standard solutions of NNAL and nicotine were prepared inphosphate buffers over the pH range 3.0-7.5. The buffered standardsolution was passed through the column, this fraction was collected andanalyzed for NNAL and nicotine content. A buffered wash solution waspassed through the column, this fraction was also collected and analyzedfor NNAL and nicotine content.

The solutions were analyzed by HPLC with UV detection. Optimumconditions for the MIP to retain NNAL and recover nicotine are observedat the pH range 4.0-4.5. At lower pH values the nicotine is protonatedand has little interaction with the polymer, so is carried through withthe aqueous buffer.

Example 12: Use of the MIP material developed for4-methylnitrosoamino-1-(3-pyridyl)-1-butanol (NNAL), in the Treatment ofa NNAL and TSNA Containing Solution

The polymer produced in accordance with the method of Example 2 isincorporated into a solid phase extraction column, and the column wasconditioned by passing through dichloromethane (DCM), methanol andfinally distilled water.

Aqueous standard solutions of NNAL and TSNAs (NAB, NAT, NNK and NNN)were acidified with glacial acetic acid to pH 3. The standard solutionwas passed through the column, followed by three glacial acetic acidsolution washes. This fraction was analyzed for NNAL and TSNA content byGC-TEA. Three washes of dichloromethane were passed through the column,this fraction was also analyzed for NNAL and TSNA content.

The MIP retained 91% of the NNAL, 65% of the NNK and an efficiency ofabout 20-30% for the other (less structurally similar) TSNAs.

The foregoing disclosure has been set forth merely to illustrate theinvention and is not intended to be limiting. Since modifications of thedisclosed embodiments incorporating the spirit and substance of theinvention may occur to persons skilled in the art, the invention shouldbe construed to include everything within the scope of the appendedclaims and equivalents thereof.

The invention claimed is:
 1. A molecularly imprinted polymer selectivefor at least one tobacco-specific nitrosamine, wherein said molecularlyimprinted polymer is obtained using a functional or structural analogueof a tobacco-specific nitrosamine as a template, and includes an imprintof the functional or structural analogue of a tobacco-specificnitrosamine.
 2. The molecularly imprinted polymer according to claim 1,wherein said at least one tobacco-specific nitrosamine is derived fromnicotine, nornicotine, anabasine or anatabine.
 3. The molecularlyimprinted polymer according to claim 1, selective for thenicotine-derived nitrosamine NNAL.
 4. The molecularly imprinted polymeraccording to claim 1, wherein the polymer has been prepared using anisosteric analogue of a tobacco-specific nitrosamine as a template. 5.The molecularly imprinted polymer according to claim 1, wherein thepolymer has been prepared using4-(methylpropenyl-amino)-1-pyridin-3-yl-butan-1-ol as a template.
 6. Themolecularly imprinted polymer according to claim 1, wherein the polymerhas been prepared using pyridine carbinol as a template.
 7. Themolecularly imprinted polymer according to claim 1, wherein the polymerhas been prepared using a compound that includes a substructure of thetobacco specific nitrosamine.
 8. A kit, comprising: the molecularlyimprinted polymer according to claim 1; and instructions for using themolecularly imprinted polymer to perform at least one of detecting,quantifying, and separating nitrosamines in a sample.
 9. A smokingarticle, comprising: a smoking material; and the molecularly imprintedpolymer according to claim 1, selective for at least onetobacco-specific nitrosamine found in the thermall decompositionproducts of the smoking material.
 10. The smoking article according toclaim 9, wherein the molecularly imprinted polymer is selective for atleast one volatile nitrosamine found in the vapor phase of the thermaldecomposition products of the smoking material.
 11. A smoke filter,comprising: filter material; and the molecularly imprinted polymeraccording to claim
 1. 12. The smoke filter according to claim 11,wherein the molecularly imprinted polymer is selective for at least onevolatile nitrosamine found in the vapor phase of the thermaldecomposition products of tobacco or a tobacco substitute.